Polypeptides Having Lipase Activity And Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having lipase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/599,199, filed Aug. 30, 2012, which is a divisional applicationof U.S. application Ser. No. 13/440,642, filed Apr. 5, 2012, which is adivisional application of U.S. application Ser. No. 13/294,047, filedNov. 10, 2011, which is a divisional of U.S. application Ser. No.13/114,258, filed May 24, 2011, now U.S. Pat. No. 8,067,218, which is adivisional application of U.S. application Ser. No. 12/944,557, filedNov. 11, 2010, now U.S. Pat. No. 7,955,830, which is a divisionalapplication of U.S. application Ser. No. 12/687,696, filed Jan. 14,2010, now U.S. Pat. No. 7,855,172, which is a divisional application ofU.S. application Ser. No. 11/255,553, filed Oct. 21, 2005, now U.S. Pat.No. 7,662,602, which claims the benefit of U.S. Provisional ApplicationNo. 60/621,282, filed Oct. 21, 2004, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having lipaseactivity and isolated polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods for producingand using the polypeptides.

2. Description of the Related Art

Triacylglycerol hydrolyzing enzymes are enzymes that catalyze thehydrolysis or formation of triglycerides. Triacylglycerol hydrolyzingenzymes are a versatile group of enzymes and often have more than oneactivity such as lipase, phospholipase, lysophospholipase, cholesterolesterase, cutinase, amidase, galactolipase, and other esterase type ofactivities. Which activity is the predominant activity will depend onthe application of the enzyme and the conditions.

Triacylglycerol hydrolyzing enzymes belong to the IUBMB EnzymeNomenclature 3.1.1. EC 3 refers to hydrolases, EC 3.1 refers to actingon ester bonds, and EC 3.1.1 refers to carboxylic ester hydrolases.Related enzymes are classified in EC 3.1.4, which refers to phosphoricdiester hydrolases.

Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of a widerange of carboxy esters, e.g., triglycerides to release fatty acid.Esterases (EC 3.1.1.1) are enzymes that catalyze the hydrolysis ofwater-soluble carboxylic esters, including short-chain fatty acidtriglycerides, to produce an alcohol and a carboxylic acid anion.

Some lipases also have phospholipase activity and/or galactolipaseactivity (see, for example, U.S. Pat. No. 6,103,505 and U.S. Pat. No.6,852,346), and can also have sterol esterase activity. (3.1.1.13).

Phospholipases are enzymes that catalyze the hydrolysis of phospholipidswhich consist of a glycerol backbone with two fatty acids in the sn1 andsn2 positions, which is esterified with a phosphoric acid in the sn3position. The phosphoric acid may, in turn, be esterified to an aminoalcohol.

There are several types of phospholipases which catalyze the hydrolysisof the fatty acyl moieties. These phospholipases include phospholipaseA₁ (EC 3.1.1.32), phospholipase A₂ (EC 3.1.1.4), and lysophospholipase(EC 3.1.1.5). Phospholipase C (EC 3.1.4.3) and phospholipase D (EC3.1.4.4) hydrolyze the phosphoric acid group from a phospholipid, but donot hydrolyze fatty acids like phospholipase A₁, phospholipase A₂ andphospholipase B.

Phospholipase A₁ (EC 3.1.1.32) catalyzes the deacylation of one fattyacyl group in the sn1 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid. Phospholipase A₂ (EC 3.1.1.4)catalyzes the deacylation of one fatty acyl group in the sn2 positionfrom a diacylglycerophospholipid to produce lysophospholipid and fattyacid. Lysophospholipase (EC 3.1.1.5), also known as phospholipase B,catalyzes the hydrolysis of the fatty acyl group in a lysophospholipid.Phospholipase C (EC 3.1.4.3) catalyzes the hydrolysis ofphosphatidylcholine to 1,2-diacylglycerol and choline phosphate.Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of the terminalphosphate diester bond of phosphatidylcholine to produce choline andphosphatidic acid.

Galactolipases (EC 3.1.1.26) catalyze the hydrolysis of galactolipids byremoving one or two fatty acids.

Sterol esterases (3.1.1.13) catalyze the hydrolysis of sterol esters tosterol and fatty acid.

Detergents formulated with lipolytic enzymes are known to have improvedproperties for removing fatty stains. For example, LIPOLASE™ (NovozymesA/S, Bagsværd, Denmark), a microbial lipase obtained from the fungusThermomyces lanuginosus (also called Humicola lanuginosa), has beenintroduced into many commercial brands of detergent. Lipases have alsobeen used in degumming processes and baking.

El-Shahed et al., 1988, Egypt. J. Microbiol. 23: 537-547 and Mohawed etal., 1988, Egypt. J. Microbiol. 23: 357-372 disclose two Aspergillusfumigatus lipases.

WO 03/12071 discloses a gene encoding a lipase from Aspergillusfumigatus.

Mayordomo et al., 2000, J. Agric. Chem. 48: 105-109 disclose theisolation, purification, and characterization of a cold-active lipasefrom Aspergillus nidulans.

Kundu et al., 1987, Journal of General Microbiology 133: 149-154,disclose the isolation and characterization of an extracellular lipasefrom the conidia of Neurospora crassa.

Lipases have many commercial uses but very few lipases that work underapplication conditions and can be produced with high yields by microbialfermentation have been identified. There is a need in the art foralternative lipases with improved properties.

It is an object of the present invention to provide polypeptides havinglipase activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having lipaseactivity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 60%identity with the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;

(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under at least medium stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or (iii) a complementarystrand of (i) or (ii); and

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

The present invention also relates to isolated polynucleotides encodingpolypeptides having lipase activity, selected from the group consistingof:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 60% identity with the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10;

(b) a polynucleotide having at least 60% identity with the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, or SEQ ID NO: 9; and

(c) a polynucleotide which hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or(iii) a complementary strand of (i) or (ii).

In a preferred aspect, the mature polypeptide is amino acids 21 to 562of SEQ ID NO: 2, amino acids 25 to 588 of SEQ ID NO: 4, amino acids 21to 598 of SEQ ID NO: 6, amino acids 19 to 534 of SEQ ID NO: 8, or aminoacids 22 to 578 of SEQ ID NO: 10. In another preferred aspect, themature polypeptide coding sequence is nucleotides 61 to 1686 of SEQ IDNO: 1, nucleotides 73 to 1764 of SEQ ID NO: 3, nucleotides 61 to 1922 ofSEQ ID NO: 5, nucleotides 55 to 1602 of SEQ ID NO: 7, or nucleotides 64to 1789 of SEQ ID NO: 9.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing such apolypeptide having lipase activity comprising: (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of using the polypeptideshaving lipase activity in detergents, degumming, and baking.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of nucleotides 1 to 60 of SEQ ID NO: 1, nucleotides 1 to 72of SEQ ID NO: 3, nucleotides 1 to 60 of SEQ ID NO: 5, nucleotides 1 to54 of SEQ ID NO: 7, or nucleotides 1 to 63 of SEQ ID NO: 9, wherein thegene is foreign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pJLin173.

FIGS. 2A and 2B show the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus lipase (lip1, SEQ ID NOs: 1 and 2,respectively).

FIG. 3 shows a restriction map of pAILo1.

FIG. 4 shows a restriction map of pBM121b.

FIG. 5 shows a restriction map of pBM120a.

FIG. 6 shows a restriction map of pJLin172.

FIG. 7 shows a restriction map of pSMO230.

FIGS. 8A and 8B shows the genomic DNA sequence and the deduced aminoacid sequence of a Fusarium graminearum NRRL 31084 lipase (SEQ ID NOs: 3and 4, respectively).

FIG. 9 shows a restriction map of pEJG61.

FIG. 10 shows a restriction map of pSMO232.

FIG. 11 shows a restriction map of pJLin175.

FIGS. 12A and 12B show the genomic DNA sequence and the deduced aminoacid sequence of a Magnaporthe grisea lipase (SEQ ID NOs: 5 and 6,respectively).

FIG. 13 shows a restriction map of pCrAm140.

FIGS. 14A and 14B show the genomic DNA sequence and the deduced aminoacid sequence of a Magnaporthe grisea lipase (SEQ ID NOs: 7 and 8,respectively).

FIG. 15 shows a restriction map of pBM134b.

FIGS. 16A and 16B shows the genomic DNA sequence and the deduced aminoacid sequence of a Neurospora crassa FGSC 2489 lipase (SEQ ID NOs: 9 and10, respectively).

DEFINITIONS

Lipase activity: The term “lipase activity” is defined herein as atriacylglycerol acylhydrolase activity (E.C. 3.1.1.3) which catalyzesthe hydrolysis of a triacylglycerol to fatty acid(s). The substratespectrum of lipases includes triglycerides, diglycerides, andmonoglycerides, but for the purpose of the present invention, lipaseactivity is determined using p-nitrophenyl butyrate as substrate. Oneunit of lipase activity equals the amount of enzyme capable of releasing1 μmole of butyric acid per minute at pH 7.5, 25° C. Encompassed withinthe term “lipase activity” are polypeptides that also have phopholipaseactivity, sterol ester esterase activity, and/or galactolipase activity,as defined herein.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the lipase activity of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,or SEQ ID NO: 10.

Phospholipase activity: The term “phospholipase activity” is definedherein as a phosphatidyl acylhydrolase (EC 3.1.1.4, EC 3.1.1.5, and EC3.1.1.32) which catalyzes the hydrolysis of a fatty acid from aphospholipid. Phospholipids consist of a glycerol backbone with twofatty acids in the sn1 and sn2 positions, which is esterified with aphosphoric acid in the sn3 position. The phosphoric acid may, in turn,be esterified with an amino alcohol.

Phospholipase A₁ (EC 3.1.1.32) catalyzes the deacylation of one fattyacyl group in the sn1 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid.

Phospholipase A₂ (EC 3.1.1.4) catalyzes the deacylation of one fattyacyl group in the sn2 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid.

Lysophospholipase (EC 3.1.1.5), also known as phospholipase B, catalyzesthe hydrolysis of the fatty acyl group in a lysophospholipid.

For purposes of the present invention, phospholipase A₁, phospholipaseA₂, and lysophospholipase activity is determined according to WO2005/040410 using phosphatidylcholines derived from soy (Avanti PolarLipids Inc., AL, USA) or alkylated phosphatidylethanolamines assubstrates.

Galactolipase activity: The term “galactolipase activity” is definedherein as a 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol acylhydrolase (EC3.1.1.26) which catalyzes the hydrolysis of galactolipids by removingone or two fatty acids. Galactolipase activity is determined accordingto WO 2005/040410 using digalactosyldiglyceride (DGDG) ormonogalactosyldiglyceride (MGDG) extracted from wheat flour assubstrate. DGDG is a galactolipid that consists of two fatty acids and adigalactose. MGDG is a galactolipid that consists of two fatty acids anda galactose.

Sterol esterase activity: The term “sterol esterase activity” is definedherein as sterol ester acylhydrolase (3.1.1.13) which catalyzes thehydrolysis of sterol esters to sterol and fatty acid, e.g., cholesterolester to cholesterol and fatty acid. Sterol esterase activity isdetermined according to WO 05/040410 using cholesterol linoleate as asubstrate.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively associated. It is, therefore,preferred that the substantially pure polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having lipase activity that is in its final form followingtranslation and any post-translational modifications, such as N-terminalprocessing, C-terminal truncation, glycosylation, etc.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”. Forpurposes of the present invention, the degree of identity between twoamino acid sequences is determined by the Clustal method (Higgins, 1989,CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR,Inc., Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein which gives an E value (or expectancy score) of lessthan 0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Thermomyces lanuginosus lipase (Accession No. 059952).

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; or a homologoussequence thereof; wherein the fragment has lipase activity. In apreferred aspect, a fragment contains at least 467 amino acid residues,more preferably at least 492 amino acid residues, and most preferably atleast 517 amino acid residues of the mature polypeptide of SEQ ID NO: 2or a homologous sequence thereof. In another preferred aspect, afragment contains at least 485 amino acid residues, more preferably atleast 510 amino acid residues, and most preferably at least 535 aminoacid residues of SEQ ID NO: 4 of the mature polypeptide of SEQ ID NO: 4or a homologous sequence thereof. In another preferred aspect, afragment contains at least 500 amino acid residues, more preferably atleast 525 amino acid residues, and most preferably at least 550 aminoacid residues of the mature polypeptide of SEQ ID NO: 6 or a homologoussequence thereof. In another preferred aspect, a fragment contains atleast 440 amino acid residues, more preferably at least 465 amino acidresidues, and most preferably at least 490 amino acid residues of themature polypeptide of SEQ ID NO: 8 or a homologous sequence thereof. Inanother preferred aspect, a fragment contains at least at least 460amino acid residues, more preferably at least 485 amino acid residues,and most preferably at least 530 amino acid residues of SEQ ID NO: 10 ora homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; or a homologoussequence thereof; wherein the subsequence encodes a polypeptide fragmenthaving lipase activity. In a preferred aspect, a subsequence contains atleast 1401 nucleotides, more preferably at least 1476 nucleotides, andmost preferably at least 1551 nucleotides of the mature polypeptidecoding sequence of SEQ ID NO: 1 or a homologous sequence thereof. Inanother preferred aspect, a subsequence contains at least 1455nucleotides, more preferably at least 1530 nucleotides, and mostpreferably at least 1605 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 3 or a homologous sequence thereof. In apreferred aspect, a subsequence contains at least 1500 nucleotides, morepreferably at least 1575 nucleotides, and most preferably at least 1650nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 5 ora homologous sequence thereof. In another preferred aspect, asubsequence contains at least 1320 nucleotides, more preferably at least1395 nucleotides, and most preferably at least 1470 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 7 or a homologoussequence thereof. In another preferred aspect, a subsequence contains atleast 1380 nucleotides, more preferably at least 1455 nucleotides, andmost preferably at least 1590 nucleotides of the mature polypeptidecoding sequence of SEQ ID NO: 9 or a homologous sequence thereof.Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having lipase activity.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other.

Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleotidesequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG and TGA. The coding sequence may be aDNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10; or a homologous sequence thereof; as well as genetic manipulation ofthe DNA encoding such a polypeptide. The modification can besubstitutions, deletions and/or insertions of one or more amino acids aswell as replacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having lipase activity produced by an organismexpressing a modified nucleotide sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, or SEQ ID NO: 9; or a homologous sequence thereof. The modifiednucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; or a homologoussequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having LipaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, of at least 60%, preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 97%, 98%, or 99%, which have lipase activity(hereinafter “homologous polypeptides”). In a preferred aspect, thehomologous polypeptides have an amino acid sequence which differs by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 2. In another preferred aspect, a polypeptide comprisesamino acids 21 to 562 of SEQ ID NO: 2, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 21 to 562 of SEQ ID NO: 2.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptideconsists of amino acids 21 to 562 of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has lipase activity. In anotherpreferred aspect, a polypeptide consists of amino acids 21 to 562 of SEQID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 4, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 4. In another preferred aspect, a polypeptide comprisesamino acids 25 to 588 of SEQ ID NO: 4, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 25 to 588 of SEQ ID NO: 4.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide consists of amino acids 25 to588 of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 25 to 588 of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 6, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 6. In another preferred aspect, a polypeptide comprisesamino acids 21 to 598 of SEQ ID NO: 6, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 21 to 598 of SEQ ID NO: 6.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide consists of amino acids 21 to598 of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 21 to 598 of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 8, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 8. In another preferred aspect, a polypeptide comprisesamino acids 19 to 534 of SEQ ID NO: 8, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 19 to 534 of SEQ ID NO: 8.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide consists of amino acids 19 to534 of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 19 to 534 of SEQ ID NO: 8.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 10, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 10.

In another preferred aspect, a polypeptide comprises amino acids 22 to578 of SEQ ID NO: 10, or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide comprises amino acids 22 to 578 of SEQ ID NO: 10. In anotherpreferred aspect, a polypeptide consists of the amino acid sequence ofSEQ ID NO: 10 or an allelic variant thereof; or a fragment thereof thathas lipase activity. In another preferred aspect, a polypeptide consistsof the amino acid sequence of SEQ ID NO: 10. In another preferredaspect, a polypeptide consists of amino acids 22 to 578 of SEQ ID NO: 10or an allelic variant thereof; or a fragment thereof that has lipaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 22 to 578 of SEQ ID NO: 10.

In a second aspect, the present invention relates to isolatedpolypeptides having lipase activity which are encoded by polynucleotideswhich hybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,(iii) a subsequence of (i) or (ii), or (iv) a complementary strand of(i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). A subsequence of the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9contains at least 100 contiguous nucleotides or preferably at least 200contiguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment which has lipase activity. In a preferred aspect,the mature polypeptide coding sequence is nucleotides 61 to 1686 of SEQID NO: 1, nucleotides 73 to 1764 of SEQ ID NO: 3, nucleotides 61 to 1922of SEQ ID NO: 5, nucleotides 55 to 1602 of SEQ ID NO: 7, or nucleotides64 to 1789 of SEQ ID NO: 9.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, or SEQ ID NO: 9; or a subsequence thereof; as well as theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10; or a fragment thereof; may be used to design anucleic acid probe to identify and clone DNA encoding polypeptideshaving lipase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength. For example, the nucleic acid probe may be at least 200nucleotides, preferably at least 300 nucleotides, more preferably atleast 400 nucleotides, or most preferably at least 500 nucleotides inlength. Even longer probes may be used, e.g., nucleic acid probes whichare at least 600 nucleotides, at least preferably at least 700nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having lipase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9; or a subsequence thereof; the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; itscomplementary strand; or a subsequence thereof; under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 61 to 1686 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pJLin173 which is contained in E. coliNRRL B-30782, wherein the polynucleotide sequence thereof encodes apolypeptide having lipase activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding region contained inplasmid pJLin173 which is contained in E. coli NRRL B-30782.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 1764 of SEQ ID NO:3. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 4,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 3. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMO230 whichis contained in E. coli NRRL B-30803, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pSMO230 which iscontained in E. coli NRRL B-30803.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 1922 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 6,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pJLin175 whichis contained in E. coli NRRL B-30783, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pJLin175 which iscontained in E. coli NRRL B-30783.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1602 of SEQ ID NO:7. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 8,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 7. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pCrAm140 whichis contained in E. coli NRRL B-30788, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pCrAm140 which iscontained in E. coli NRRL B-30788.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1789 of SEQ ID NO:9. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 10,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 9. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pBM134b whichis contained in E. coli NRRL B-30786, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pBM134b which iscontained in E. coli NRRL B-30786.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(n), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; or a homologoussequence thereof. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,lipase activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 21 to 562 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 4, such as aminoacids 25 to 588 of SEQ ID NO: 4, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 6, such as aminoacids 21 to 598 of SEQ ID NO: 6, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 8, such as aminoacids 19 to 534 of SEQ ID NO: 8, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 10, such as aminoacids 22 to 578 of SEQ ID NO: 8, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Lipase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide having lipase activity, e.g.,a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide having lipase activity; or a Streptomyces polypeptide havinglipase activity, e.g., a Streptomyces lividans or Streptomyces murinuspolypeptide having lipase activity; or a gram negative bacterialpolypeptide having lipase activity, e.g., an E. coli or a Pseudomonassp. polypeptide having lipase activity.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide having lipase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having lipaseactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide having lipaseactivity.

In another preferred aspect, the polypeptide is a Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, Thielavia terrestris, Thielavia terricola, Thielaviathermophila, Thielavia variospora, or Thielavia wareingii polypeptidehaving lipase activity.

In a more preferred aspect, the polypeptide is an Aspergillus fumigatuspolypeptide, e.g., the polypeptide of SEQ ID NO: 2, or the maturepolypeptide thereof.

In another more preferred aspect, the polypeptide is a Fusariumgraminearum polypeptide, and most preferably Fusarium graminearum NRRL31084, e.g., the polypeptide of SEQ ID NO: 4, or the mature polypeptidethereof.

In another more preferred aspect, the polypeptide is a Magnaporthegrisea polypeptide, and most preferably Magnaporthe grisea FGSC 8958(Fungal Genetics Stock Center, Kansas City, Mo.) polypeptide, e.g., thepolypeptide of SEQ ID NO: 6 or SEQ ID NO: 8; or the mature polypeptidethereof.

In another more preferred aspect, the polypeptide is an Aspergillusnidulans polypeptide, and most preferably Aspergillus nidulans FGSC 2489(Fungal Genetics Stock Center, Kansas City, Mo.) polypeptide, e.g., thepolypeptide of SEQ ID NO: 10; or the mature polypeptide thereof.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.

In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is thesequence contained in plasmid pJLin173 which is contained in E. coliNRRL B-30782. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 1. In anotherpreferred aspect, the nucleotide sequence is nucleotides 61 to 1686 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pJLin173which is contained in E. coli NRRL B-30782. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,which differ from SEQ ID NO: 1 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 1 which encodefragments of SEQ ID NO: 2 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 3. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pSMO230 which is contained in E. coliNRRL B-30803. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 3. In anotherpreferred aspect, the nucleotide sequence is nucleotides 73 to 1764 ofSEQ ID NO: 3. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pSMO230which is contained in E. coli NRRL B-30803. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 4 or the mature polypeptide thereof,which differ from SEQ ID NO: 3 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 3 which encodefragments of SEQ ID NO: 4 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 5. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pJLin175 which is contained in E. coliNRRL B-30783. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 5. In anotherpreferred aspect, the nucleotide sequence is nucleotides 61 to 1922 ofSEQ ID NO: 5. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pJLin175which is contained in E. coli NRRL B-30783. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 6 or the mature polypeptide thereof,which differ from SEQ ID NO: 5 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 5 which encodefragments of SEQ ID NO: 6 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 7. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pCrAm140 which is contained in E. coliNRRL B-30788. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 7. In anotherpreferred aspect, the nucleotide sequence is nucleotides 55 to 1602 ofSEQ ID NO: 7. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pCrAm140which is contained in E. coli NRRL B-30788. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof,which differ from SEQ ID NO: 7 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 7 which encodefragments of SEQ ID NO: 8 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 9. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pBM134b which is contained in E. coliNRRL B-30786. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 9. In anotherpreferred aspect, the nucleotide sequence is nucleotides 64 to 1789 ofSEQ ID NO: 9. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pBM134bwhich is contained in E. coli NRRL B-30786. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 10 or the mature polypeptide thereof,which differ from SEQ ID NO: 9 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 9 which encodefragments of SEQ ID NO: 10 that have lipase activity.

The present invention also relates to mutant polunucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 2. In a preferred aspect, the maturepolypeptide is amino acids 21 to 562 of SEQ ID NO: 2.

The present invention also relates to mutant polunucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 3, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 4. In a preferred aspect, the maturepolypeptide is amino acids 25 to 588 of SEQ ID NO: 4.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 5, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 6. In a preferred aspect, the maturepolypeptide is amino acids 21 to 598 of SEQ ID NO: 6.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 7, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 8. In a preferred aspect, the maturepolypeptide is amino acids 19 to 534 of SEQ ID NO: 8.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 9, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 10. In a preferred aspect, the maturepolypeptide is amino acids 22 to 578 of SEQ ID NO: 10.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thielavia, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 of at least 60%, preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, even more preferably at least 95%, and most preferably atleast 97% identity, which encode an active polypeptide. In a preferredaspect, the mature polypeptide coding sequence is nucleotides 61 to 1686of SEQ ID NO: 1.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 3 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 73 to 1764 of SEQ ID NO: 3.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 5 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 61 to 1922 of SEQ ID NO: 5.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 7 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 55 to 1602 of SEQ ID NO: 7.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 9 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 64 to 1789 of SEQ ID NO: 9.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,e.g., a subsequence thereof, and/or by introduction of nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe polypeptide encoded by the nucleotide sequence, but which correspondto the codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions which may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forlipase activity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or (iii) a complementary strand of(i) or (ii); or allelic variants and subsequences thereof (Sambrook etal., 1989, supra), as defined herein.

In a preferred aspect, the mature polypeptide coding sequence of SEQ IDNO: 1 is nucleotides 61 to 1686. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 3 is nucleotides 73 to 1764.In another preferred aspect, the mature polypeptide coding sequence ofSEQ ID NO: 5 is nucleotides 61 to 1922. In another preferred aspect, themature polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 55 to1602. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 9 is nucleotides 64 to 1789.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or (iii) a complementarystrand of (i) or (ii); and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having lipase activity. In a preferredaspect, the mature polypeptide coding sequence of SEQ ID NO: 1 isnucleotides 61 to 1686. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 3 is nucleotides 73 to 1764.In another preferred aspect, the mature polypeptide coding sequence ofSEQ ID NO: 5 is nucleotides 61 to 1922. In another preferred aspect, themature polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 55 to1602. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 9 is nucleotides 64 to 1789.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 60 of SEQ ID NO: 1 which encode amino acids 1 to 20 of SEQ ID NO: 2.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 72 of SEQ ID NO: 3 which encode amino acids 1 to 24 ofSEQ ID NO: 4.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 60 of SEQ ID NO: 5 which encode amino acids 1 to 20 ofSEQ ID NO: 6.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 54 of SEQ ID NO: 7 which encode amino acids 1 to 18 ofSEQ ID NO: 8.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 63 of SEQ ID NO: 9 which encode amino acids 1 to 21 ofSEQ ID NO: 10.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. In a preferred aspect,the cell is of the genus Aspergillus. In a more preferred aspect, thecell is Aspergillus fumigatus. In another more preferred aspect, thecell is Aspergillus nidulans. In another preferred aspect, the cell isof the genus Magnaporthe. In another more preferred aspect, the cell isMagnaporthe grisea.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,wherein the mutant nucleotide sequence encodes a polypeptide whichcomprises or consists of the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, and (b) recoveringthe polypeptide.

In a preferred aspect, the mature polypeptide of SEQ ID NO: 2 is aminoacids 21 to 562. In another preferred aspect, the mature polypeptide ofSEQ ID NO: 4 is amino acids 25 to 588. In another preferred aspect, themature polypeptide of SEQ ID NO: 6 is amino acids 21 to 598. In anotherpreferred aspect, the mature polypeptide of SEQ ID NO: 8 is amino acids19 to 534. In another preferred aspect, the mature polypeptide of SEQ IDNO: 10 is amino acids 22 to 578.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having lipase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having lipase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Lipase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity byfermentation of a cell which produces both a polypeptide of the presentinvention as well as the protein product of interest by adding aneffective amount of an agent capable of inhibiting lipase activity tothe fermentation broth before, during, or after the fermentation hasbeen completed, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity bycultivating the cell under conditions permitting the expression of theproduct, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the lipase activity substantially,and recovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with an lipase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the lipase activity. Complete removal of lipase activity may beobtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallylipase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thelipase-deficient cells may also be used to express heterologous proteinsof pharmaceutical interest such as hormones, growth factors, receptors,and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from lipase activity which is produced by a method ofthe present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thelipase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having lipase activity, or compositions thereof.

Use in Degumming.

A polypeptide of the present invention may be used for degumming anaqueous carbohydrate solution or slurry to improve its filterability,particularly, a starch hydrolysate, especially a wheat starchhydrolysate which is difficult to filter and yields cloudy filtrates.The treatment may be performed using methods well known in the art. See,for example, EP 219,269, EP 808,903, and U.S. Pat. No. 6,103,505.

Use in Baking.

A polypeptide of the present invention may be used in baking accordingto U.S. Pat. No. 6,558,715.

Use in detergents.

The polypeptides of the present invention may be added to and thusbecome a component of a detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the present invention as describedherein. The detergent additive as well as the detergent composition maycomprise one or more enzymes such as a protease, lipase, cutinase, anamylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™ Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™ Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include Lipolase™,Lipolase Ultra™, and Lipex™ (Novozymes A/S).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Amylases include, for example, α-amylases obtained from Bacillus, e.g.,a special strain of Bacillus licheniformis, described in more detail inGB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a polypeptide of the present inventionmay be added in an amount corresponding to 0.001-100 mg of protein,preferably 0.005-50 mg of protein, more preferably 0.01-25 mg ofprotein, even more preferably 0.05-10 mg of protein, most preferably0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein perliter of wash liquor.

A polypeptide of the present invention may also be incorporated in thedetergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a nucleotide sequencecomprising or consisting of nucleotides 1 to 60 of SEQ ID NO: 1,nucleotides 1 to 72 of SEQ ID NO: 3, nucleotides 1 to 60 of SEQ ID NO:5, nucleotides 1 to 54 of SEQ ID NO: 7, or nucleotides 1 to 63 of SEQ IDNO: 9, encoding a signal peptide comprising or consisting of amino acids1 to 20 of SEQ ID NO: 2, amino acids 1 to 24 of SEQ ID NO: 4, aminoacids 1 to 20 of SEQ ID NO: 6, amino acids 1 to 18 of SEQ ID NO: 8, oramino acids 1 to 21 of SEQ ID NO: 10, respectively, wherein the gene isforeign to the nucleotide sequence.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus fumigatus PaHa34, Fusarium graminearum NRRL 31084,Magnaporthe grisea FGSC 8958 (Fungal Genetics Stock Center), andAspergillus nidulans FGSC 2489 (Fungal Genetics Stock Center) were usedas the sources of the lipase genes.

Media

SOC medium was composed per liter of 20 g of tryptone, 5 g of yeastextract, 2 ml of 5 M NaCl, and 2.5 ml of 1 M KCl.

NZY⁺ medium was composed per liter of 10 g of NZ amine, 5 g of yeastextract, 5 g of NaCl, 12.5 mM MgCl₂, 12.5 mM MgSO₄, and 10 ml of 2 Mglucose.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl.

MY25 medium was composed per liter of 25 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g ofurea, 10 g of yeast extract, and 1.5 ml of AMG trace metals solution,adjusted to pH 6.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

COVE selection plates were composed per liter of 342.3 g of sucrose, 20ml of COVE salt solution, 10 mM acetamide, 15 mM CsCl₂, and 25 g ofNoble agar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE trace metals solution was composed per liter of 0.04 g ofNaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

2×YT plates were composed per liter of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl, and 15 g of Bacto agar.

M400 medium was composed per liter of 50 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 2 g of KH₂PO₄, 4 g of citric acid, 8 g of yeast extract, 2 gof urea, 0.5 g of CaCl₂, and 0.5 ml of AMG trace metals solution.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

NZY⁺ medium was composed per liter of 10 g of NZ amine, 5 g of yeastextract, 5 g of NaCl, 12.5 mM MgCl₂, 12.5 mM MgSO₄, and 10 ml of 2 Mglucose.

RA sporulation medium was composed per liter of 1 g of glucose, 50 g ofsuccinic acid, 12.1 g of NaNO₃, and 20 ml of 50× Vogel's salts (no C, noN).

50× Vogel's salts (no C, no N) was composed per liter of 250 g ofKH₂PO₄, 10 g of MgSO₄.7H₂O, 5 g of CaCl₂.2H₂O, 2.5 ml of biotinsolution, and 5 ml of Vogel's trace elements.

50× Vogel's trace elements solution was composed per liter of 50 g ofcitric acid, 50 g of ZnSO₄.7H₂O, 10 g of Fe(NH₄)₂(SO₄)₂.6H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of MnSO₄.H₂O, 0.5 g of H₃BO₃, and 0.5 g ofNa₂MoO₄.2H₂O.

YPG medium was composed per liter of 1% yeast extract, 2% bactopeptone,and 5% glucose.

Vogel's NO₃ regeneration low-melt medium was composed per liter of 20 mlof 50× Vogels solution with 25 mM NaNO₃ stock, 0.8 M sucrose and 1.5%low melting agarose (Sigma Chemical Company, St. Louis, Mo.). WhereBASTA™ was added to the medium, BASTA™ was obtained from AgrEvo (HoechstSchering, Rodovre, Denmark) and was extracted twice withphenol:chloroform:isoamyl alcohol (25:24:1), and once withchloroform:isoamyl alcohol (24:1) before use.

50× Vogels solution with 25 mM NaNO₃ stock was composed of per liter of125 g of sodium citrate, 250 g of KH₂PO₄, 106.25 g of NaNO₃, 10 g ofMgSO₄.7H₂O, 5 g of CaCl₂.2H₂O, 2.5 g of biotin solution, and 5 ml of 50×Vogels trace element solution.

Biotin stock solution was composed of 5 mg of biotin in 100 ml of 50%ethanol.

200×AMG trace metals solution was composed per liter of 3 g of citricacid, 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 13.8 g of FeSO₄.7H₂O,and 8.5 g of MnSO₄.H₂O.

STC was composed of 0.8 M sorbitol, 50 mM CaCl₂, and 25 mM Tris-Cl, pH8.

SPTC was composed of 0.8 M sorbitol, 25 mM Tris-HCl, pH 8, 50 mM CaCl₂,and 40% PEG 4000.

CM medium was composed per liter of 6 g yeast extract, 6 g casein acidhydrolysate, and 10 g sucrose.

Example 1 Identification of Lipase Genes in the Partial Genomic Sequenceof Aspergillus fumigatus

A tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods andProtocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was carried out using as query a lipasesequence from Geotrichum candidum (SWALL P17577). Several genes wereidentified as putative lipases based upon a high degree of similarity tothe query sequence at the amino acid level. A genomic region ofapproximately 1562 bp with greater than 39% identity to the querysequence at the amino acid level was chosen for further study. Genemodels for the putative lipase genes were predicted based on homology tothe Geotrichum candidum lipase as well as conserved sequences present atthe 5′ and 3′ ends of fungal introns.

Example 2 Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in 10 mM Tris-1 mM EDTA (TE), and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, which was resuspended in pH 8.0 buffer containing 10 mMTris, 100 mM EDTA, 1% Triton X-100, 0.5 M guanidine-HCl, and 200 mMNaCl. DNase-free RNase A was added at a concentration of 20 mg/liter andthe lysate was incubated at 37° C. for 30 minutes. Cellular debris wasremoved by centrifugation, and DNA was isolated using a QIAGEN Maxi 500column (QIAGEN Inc., Valencia, Calif.). The columns were equilibrated in10 ml of QBT, washed with 30 ml of QC, and eluted with 15 ml of QF (allbuffers from QIAGEN Inc., Valencia, Calif.). DNA was precipitated inisopropanol, washed in 70% ethanol, and recovered by centrifugation. TheDNA was resuspended in TE buffer.

Example 3 Cloning of the Aspergillus fumigatus Lipase Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus fumigatus gene encoding a lipase from the genomicDNA prepared in Example 2.

Forward primer: (SEQ ID NO: 11)5′- ACACAACTGGCCATGCATCTCCTCCGGGTTGTTCTG-3′ Reverse primer:(SEQ ID NO: 12) 5′- AGTCACCTCTAGTTAATTAACTAGATATGGAACGAATCCA-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a (see Example 6).

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System (Roche Diagnostics, Mannheim, Germany). One μM ofeach of the primers above were used in a PCR reaction containing 200 ngof Aspergillus fumigatus genomic DNA, 1×PCR buffer (Roche Diagnostics,Mannheim, Germany) with 1.5 mM MgCl₂, 1 μl of dNTP mix (10 mM each), and0.75 μl of DNA polymerase mix (3.5 U/μl; Roche Diagnostics, Mannheim,Germany) in a final volume of 50 μl. To amplify the fragment, anEppendorf Mastercycler thermocycler (Hamburg, Germany) was programmedfor 1 cycle at 94° C. for 2 minutes; 10 cycles each at 94° C. for 15seconds, 61° C. for 30 seconds, and 72° C. for 1.25 minutes; 15 cycleseach at 94° C. for 15 seconds, 61° C. for 30 seconds, and 72° C. for1.25 minutes plus a 5 second elongation at each successive cycle; 1cycle at 72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 0.7% agarose gel using 44 mMTris Base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer and the 1.7 kbproduct band was purified using a QIAquick PCR Purification Kit (QIAGENInc., Valencia, Calif.) according to the manufacturer's instructions.The PCR fragment and pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) wereligated using conditions specified by the manufacturer resulting inplasmid pJLin173 (FIG. 1).

Two μl of the reaction was used to transform E. coli TOP10 One Shotcompetent cells (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. A 2 μl volume of the ligation mixture wasadded to the E. coli cells and incubated on ice for 5 minutes.Subsequently, the cells were heat shocked for 30 seconds at 42° C., andthen placed on ice for 2 minutes. A 250 μl volume of SOC medium wasadded to the cells and the mixture was incubated for 1 hour at 37° C.and 250 rpm. After the incubation the colonies were spread on 2×YTplates supplemented with 100 μg of ampicillin per ml and incubated at37° C. overnight for selection of the plasmid. Twelve colonies that grewon the plates were picked with a sterile toothpick and grown overnightat 37° C., 250 rpm in a 15 ml Falcon tube containing 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. An E. coli transformantcontaining the pJLin173 was detected by restriction digestion andplasmid DNA was prepared using a BioRobot 9600 (QIAGEN Inc., Valencia,Calif.).

E. coli TOP 10 One Shot cells containing pJLin173 were deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30782, with a deposit date of Oct. 12, 2004.

Example 4 Characterization of the Aspergillus fumigatus Genomic SequenceEncoding Lipase

DNA sequencing of the Aspergillus fumigatus lipase gene from pJLin173was performed with an Applied Biosystems Model 377 XL DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. Nucleotide sequence datawere scrutinized for quality and all sequences were compared to eachother with assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to theGeotrichum candidum lipase as well as conserved sequences present at the5′ and 3′ ends of fungal introns. A comparative alignment of amino acidsequences was made using the MAFFT method with iterative refinement anddefault parameters (Katoh et al., 2002, Nucleic Acids Research 30:3059). The nucleotide sequence (SEQ ID NO: 1) and deduced amino acidsequence (SEQ ID NO: 2) are shown in FIGS. 2A and 2B. The genomicfragment encodes a polypeptide of 562 amino acids. The % G+C content ofthe gene is 59.7% and of the mature protein coding region (nucleotides61 to 1686 of SEQ ID NO: 1) is 59.6%. Using the SignalP software program(Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of20 residues was predicted. The predicted mature protein contains 542amino acids with a molecular mass of 58.7 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=1,gap penalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus fumigatus lipase geneshared 35% identity to the deduced amino acid sequence of a Geotrichumcandidum lipase gene (SWALL P17573).

Example 5 Construction of pAILo1 expression vector

Expression vector pAILo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). Modification of pBANe6 was performedby first eliminating three Nco I restriction sites at positions 2051,2722, and 3397 bp from the amdS selection marker by site-directedmutagenesis. All changes were designed to be “silent” leaving the actualprotein sequence of the amdS gene product unchanged. Removal of thesethree sites was performed simultaneously with a GeneEditor Site-DirectedMutagenesis Kit (Promega, Madison, Wis.) according to the manufacturer'sinstructions using the following primers (underlined nucleotiderepresents the changed base):

AMDS3NcoMut (2050): (SEQ ID NO: 13) 5′-GTGCCCCATGATACGCCTCCGG-3′AMDS2NcoMut (2721): (SEQ ID NO: 14) 5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′AMDS1NcoMut (3396): (SEQ ID NO: 15) 5′-GGAGGCCATGAAGTGGACCAACGG-3′

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange MutagenesisKit (Stratagene, La Jolla, Calif.), to eliminate the Nco I restrictionsite at the end of the AMG terminator at position 1643. The followingprimers (underlined nucleotide represents the changed base) were usedfor mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 16)5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG-3′Lower Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 17)5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG-3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQuickChange Mutagenesis Kit and the following primers (underlinednucleotides represent the changed bases) to yield pAILo1 (FIG. 3).

Upper Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 18)5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 19)5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

Example 6 Construction of pBM120a Expression Vector

Plasmid pBM120a was constructed to obtain a plasmid containing thedouble NA2 promoter (NA2-NA2-tpi) for driving gene expression inAspergillus species, and containing the ampicillin resistance gene forselection in E. coli.

Primers were designed to PCR amplify the double NA2 promoter frompJaL721 (WO 03/008575). Restriction enzyme sites Sal I and Nco I(underlined) were added for cloning the double promoter into theAspergillus expression plasmid pAILo1.

(SEQ ID NO: 20) 5′-GTCGACATGGTGTTTTGATCATTTTA-3′ (SEQ ID NO: 21)5′-CCATGGCCAGTTGTGTATATAGAGGA-3′

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. The PCR amplification reaction mixture contained 1μl of 0.09 μg of pJaL721 per μl, 1 μl of each of the primers (50μmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μl of a dATP, dTTP,dGTP, and dCTP mix (10 mM each), 37.25 μl of water, and 0.75 μl of DNApolymerase mix (3.5 U/μl). To amplify the fragment, an EppendorfMastercycler thermocycler was programmed for 1 cycle at 94° C. for 2minutes; 10 cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds,and 72° C. for 1.25 minutes; 15 cycles each at 94° C. for 15 seconds,55° C. for 30 seconds, and 72° C. for 1.25 minutes plus a 5 secondelongation at each successive cycle; 1 cycle at 72° C. for 7 minutes;and a 10° C. hold. Ten microliters of this PCR reaction was mixed with 1μl of 10×DNA loading dye (25% glycerol, 10 mM Tris pH 7.0, 10 mM EDTA,0.025% bromophenol blue, 0.025% xylene cyanol) and run on a 1.0% (w/v)agarose gel using TBE buffer. The 1128 bp PCR product was observed withUV light on a Nucleotech gel visualization system (Nucleotech, SanMateo, Calif.). The PCR product was directly ligated into pPC2.1-TOPOaccording to the manufacturer's instructions. A 1 μl volume of fresh PCRproduct, 3 μl of double-distilled water, and 1 μl of the TOPO cloningvector were mixed with a pipette and incubated at room temperature for 5minutes.

After the incubation, 2 μl of the mixture was used to transform OneShotcompetent E. coli cells. A 2 μl volume of the ligation mixture was addedto the E. coli cells and incubated on ice for 5 minutes. Subsequently,the cells were heat shocked for 30 seconds at 42° C., and then placed onice for 2 minutes. A 250 μl volume of SOC medium was added to the cellsand the mixture was incubated for 1 hour at 37° C. and 250 rpm. Afterthe incubation the colonies were spread on 2×YT plates supplemented with100 μg of ampicillin per ml and incubated at 37° C. overnight forselection of the plasmid. Eight colonies that grew on the plates werepicked with a sterile toothpick and grown overnight at 37° C., 250 rpmin a 15 ml Falcon tube containing 3 ml of LB medium supplemented with100 μg of ampicillin per ml. The plasmids were isolated using a QIAGENBioRobot 9600.

Four μl volumes of the resulting plasmid minipreps were digested withEco RI. The digestion reactions were analyzed by agarose gelchromatography and UV analysis as previously described for the PCRreaction. Isolated plasmids containing an insert were sequenced using 1μl of plasmid template, 1.6 ng of M13 primer (forward or reverse) (MWGBiotech; High Point; NC), and water to 6 μl. DNA sequencing wasperformed with an Applied Biosystems Model 377 Sequencer XL (AppliedBiosystems, Inc., Foster City, Calif.) using dye-terminator chemistry.The resulting plasmid was designated pBM121b (FIG. 4).

A 5 μl volume of pBM121b was digested with Sal I and Nco I. Thedigestion reactions were analyzed by agarose gel electrophoresis asdescribed above, and ligated to the vector pAILo1, which had beenpreviously digested with Sal I and Nco I. The resulting expressionplasmid was designated pBM120a (FIG. 5).

Example 7 Construction of an Aspergillus oryzae Expression VectorExpressing Aspergillus fumigatus Lipase Gene

The 1.7 kb PCR fragment (Example 3) containing the Aspergillus fumigatuslipase gene was cloned into pBM120a using an InFusion Cloning Kit (BDBiosciences, Palo Alto, Calif.) where the vector was digested with Nco Iand Pac I. The digested vector was purified by gel electrophoresis usinga 0.7% agarose gel with TBE buffer, and the PCR fragment was extractedusing a QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) andpurified using a QIAquick PCR Purification Kit. The gene fragment andthe digested vector were ligated together in a reaction resulting in theexpression plasmid pJLin172 (FIG. 6). The ligation reaction (50 μl) wascomposed of 1× InFusion Buffer (BD Biosciences, Palo Alto, Calif.),1×BSA (BD Biosciences, Palo Alto, Calif.), 1 μl of Infusion enzyme(diluted 1:10) (BD Biosciences, Palo Alto, Calif.), 100 ng of pBM120adigested with Nco I and Pac I, and 50 ng of the Aspergillus fumigatuslipase gene purified PCR product. The reaction was incubated at roomtemperature for 30 minutes. Two μl of the reaction was used to transformE. coli SoloPack Gold supercompetent cells (Stratagene, La Jolla,Calif.) according to the manufacturer's instructions. One μl of3-mercaptoethanol was added to competent cells, and incubated on ice for10 minutes. A 2 μl volume of the ligation mixture was then added to theE. coli cells and incubated on ice for 30 minutes. Subsequently, thecells were heat shocked for 60 seconds at 54° C., and then placed on icefor 2 minutes. A 150 μl volume of NZY⁺ medium at 42° C. was added to thecells and the mixture was incubated for 1 hour at 37° C. and 250 rpm.After the incubation the colonies were spread on 2×YT platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight for selection of the plasmid. Twelve colonies that grew on theplates were picked with a sterile toothpick and grown overnight at 37°C., 250 rpm in a 15 ml Falcon tube containing 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. An E. coli transformantcontaining the pJLin172 plasmid was detected by restriction digestionand plasmid DNA was prepared using a QIAGEN BioRobot 9600.

Example 8 Expression of the Aspergillus fumigatus Lipase Gene inAspergillus oryzae BECh2

Aspergillus oryzae BECh2 (Δalp, Δamy, CPA-, KA-, Δnp1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Five μg of pJLin172 was used to transformAspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pJLin172 yielded 49transformants. The transformants were isolated to individual Coveplates. Confluent Cove plates of 35 transformants were washed with 4 mlof 0.01% Tween 20. Two hundred μl of spore suspension was inoculatedseparately into 25 ml of MY25 medium in 125 ml plastic shake flasks andincubated at 34° C., 250 rpm. Three and five days after incubation,culture supernatants were removed for lipase assay and SDS-PAGEanalysis.

Lipase activity was determined as follows: 100 μl of substrate (3.92 mlof 100 mM MOPS pH 7.5, 4 mM CaCl₂, 990 μl of DMSO, 80 μl of 1% AOS, and20 μl of p-nitrophenyl butyrate) was added to 100 μl of diluted sample.The samples were diluted accordingly in 100 mM MOPS pH 7.5, 4 mM CaCl₂.The absorbance at 405 nm was monitored for 3 minutes at room temperature(25° C.) in a 96-well microtiter plate using a Spectra MAX plate reader(Molecular Devices, Sunnyvale, Calif.).

The lipase assay results indicated that at both 3 and 5 days, 34 of the35 transformants produced lipase activity well above that of theuntransformed control. SDS-PAGE (BioRad Criterion 10-20% SDS-PAGE)analysis of 10 μl of the supernatants showed a major band atapproximately 59 kDa.

Example 9 Determination of Substrate Specificity of RecombinantAspergillus fumigatus Lipase

The substrate specificity of Aspergillus fumigatus lipase was determinedusing a panel screen composed of 4-nitrophenol (PNP) lipase substrates.

A panel screen composed of a set of 12 assays utilizing various4-nitrophenol (PNP) lipase substrates was prepared as described in theTable 1.

TABLE 1 Panel screen of PNP substrates and buffer conditions 1 mM PNP-50 mM 50 mM 50 mM PNP tagged Shorthand MOPS CHES MOPS 10 mM TritonConversion substrate designation pH 7.0 pH 9.5 pH 7.5 CaCl₂ X-100Factors 1 Palmitate 16:0 x x 1.2% 4.466 2 Palmitate 16:0 x x 1.2% 1.1 3Palmitate 16:0 x x 1.2% 2.037 4 Palmitate 16:0 x x 0.2% 1.0 5 Palmitate16:0 x x 0.2% 1.495 6 Decanoate 10:0 x x 1.2% 4.466 7 Decanoate 10:0 x x1.2% 1.1 8 Decanoate 10:0 x x 1.2% 2.037 9 Decanoate 10:0 x x 0.2% 1.010 Valerate  5:0 x x 0.40%  1.630 11 Valerate  5:0 x x   0% 1.370 12Butyrate  4:0 x x 0.40%  1.630

These assays were run in 384-well plates using 8 different dilutions ofeach sample (7 μl) to be evaluated and 80 μl of the substrates. Theassays were incubated for up to 24 hours at ambient temperature. Assayswere read at 405 nm at time points of approximately 1, 2, 3, 5, and 24hours. The results were calculated as OD/hour for each individual assay.In order to make an accurate evaluation of the amount of PNP released,it was necessary to mathematically normalize raw OD values by using aconversion factor. The conversion factors were values, determinedexperimentally, that were necessary to compensate for the fact that PNPhas lower OD readings at low pH and in the presences of detergent(Triton X100) than at pH 9.5. The factor normalizes the data to the ODreading that would have been obtained were it possible to quench thereactions to yield maximal OD for each condition while also stopping thereaction at that time point; i.e., PNP-fatty acid substrates are notstable at high pH, the tag comes off without lipase present at high pH,and the tagged substrate is particularly unstable above pH 9 and forshorter chain length fatty acid substrates.

In Table 1 the top two rows (1 and 2) were the assays used for the pHratio (9.5/7.0). Rows 3 and 10 were used for the “long chain (Slu)/shortchain (Lu); comparisons.

In Table 1 the top two rows (1 and 2) were the assays used for the pHratio (9.5/7.0). Rows 3 and 10 were used for the “long chain (Slu)/shortchain (Lu); comparisons.

LIPEX™, a Thermomyces lanuginosus lipase obtained from Novozymes A/S,Bagsværd, Denmark was used for comparison purposes.

The results for the panel screen are shown in Table 2.

TABLE 2 Ratio of PNP-substrates according to Table 1, Example 9, where:P = Palmitate, D = Decanoate, V = Valerate and B = Butyrate Longchain/Short chain at pH 7.5 P/V D/V P/B D/B A. fumigatus 0.04 0.15 0.050.16 lipase LIPEX ™ 0.997 1.944 2.122 4.080 pH Ratios; (−) = 0.2% Tritondata 9.5P/7.0P 9.5D/7.0D 9.5P−/7.5P− A. fumigatus 0.36 0.22 Not Testedlipase LIPEX ™ 2.072 2.574 2.251 No or Low Triton (−) compared tomaximum Triton (+) V7.5−/+ D9.5+/− P7.5+/− P9.5+/− A. fumigatus Not NotNot Tested Not Tested lipase Tested Tested LIPEX ™ 7.108 5.110 9.3318.166 The data for the Aspergillus fumigatus lipase was generated in96-well plate format using approximately 3 times the sample andsubstrate volumes as described in Table 1. Aspergillus fumigatus lipasetest data consists of the average of 2 assay results; and LIPEX ™ dataconsists of the average of a minimum of 80 assay results.

In comparing the panel screen results of LIPEX™ and the Aspergillusfumigatus lipase the following observations were made:

1. The ratio of activities on PNP-palmitate at pH 9.5 versus pH 7 is6-fold lower for the Aspergillus fumigatus lipase versus LIPEX™suggesting that the Aspergillus fumigatus lipase has lower activity atpH 9.5 versus LIPEX™ or it has a higher activity at pH 7.0 than LIPEX™or a combination of these two.

2. The ratios of PN, DN, and D/B are also quite different for theAspergillus fumigatus lipase versus LIPEX™ suggesting there is some acylchange length specificity differences between the two lipases.

Example 10 Purification and Characterization of Recombinant Aspergillusfumigatus Lipase

One of the Aspergillus oryzae transformants producing the highest yieldof Aspergillus fumigatus lipase (Example 8) was grown in 500 ml of MY25medium for 4 days at 30° C., 250 rpm for purification. Supernatant wassterile filtered under pressure using SEITZ-EKS filters (PALLCorporation, Waldstetten, Germany). The sterile filtered supernatant wasdiluted with distilled water so the conductivity was under 4 mSi abdthen the pH was adjusted to 7.

Source Q™ (Pharmacia Amersham, Uppsala, Sweden) was packed into a 50 mlcolumn and then washed and equilibrated with 50 mM borate pH 7 buffer.Filtered fermentation supernatant was then applied to the column usingan Akta Explorer System (Pharmacia Amersham, Uppsala, Sweden). Unboundmaterial was washed with 50 mM borate pH 9 buffer. The bound proteinswere eluted with 50 mM borate pH 9 buffer containing 0.5 M sodiumchloride as an eluent. The total length of the gradient wasapproximately 20 column volumes.

Fractions of 10 ml were collected and analyzed for lipase activityaccording to the assay described by Svendsen et al., in Methods inEnzymology, Lipases Part a Biotechnology Vol. 284 pages 317-340 Editedby Byron Rubin and Edward A. Dennis, Academic Press, 1997, New York.Aliquots of fractions were analyzed for purity by standard SDS-PAGEprocedure using Novex 4-20% Tris-glycine gels (Invitrogen LifeTechnologies, Carlsbad Calif.). The most pure fractions were pooled onthe basis of LU activity and purity assessed by SDS-PAGE. The purity ofthe pooled fractions by SDS-PAGE was determined to be more than 95% witha molecular of approximately 68 kDa.

Substrate specificity of the Aspergillus fumigatus lipase was evaluatedat pH 7 according to WO 2005/040410. The results showed that theAspergillus fumigatus lipase efficiently degrades trilinolein andcholesterol linoleate whereas activity toward phospholipids such aslecithin (and alkylated phosphatidylethanolamines) is much lower.

Example 11 Determination of Thermostability of Recombinant Aspergillusfumigatus Lipase

The thermostability of the purified recombinant Aspergillus fumigatuslipase (Example 10) was determined by Differential Scanning calorimetry(DSC). The thermal denaturation temperature, Td, was taken as the top ofthe denaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating of enzyme solutions at a constant programmedheating rate. Cp refers to heat capacity (at constant pressure). Trefers to temperature.

A VP-DSC Differential Scanning calorimeter (MicroCal Inc., Northampton,Mass.) was used for the thermostability determination according to themanufacturer's instructions. Sample enzyme and reference solutions werecarefully degassed immediately prior to loading of samples into thecalorimeter (reference: buffer without enzyme). Sample enzyme(approximately 1 mg/ml) and reference solutions (approximately 0.5 ml)were thermally pre-equillibrated for 20 minutes at 5° C. The DSC scanwas performed from 5° C. to 95° C. at a scan rate of approximately 90K/hr. Denaturation temperatures were determined at an accuracy ofapprox. +/−1° C.

The results as shown in Table 3 indicated that the Aspergillus fumigatuslipase had thermal denaturation temperatures of 60° C. in 50 mM acetatepH 5.0 buffer, 54° C. in 50 mM HEPES pH 7.0 buffer, and 44° C. in 50 mMglycine pH 10.0 buffer.

TABLE 3 Thermostability Determination Buffer pH Td (° C.) 50 mM Acetate5.0 60 50 mM HEPES 7.0 54 50 mM Glycine 10.0 44

Example 12 Identification of Lipase Genes in the Partial GenomicSequence of Fusarium graminearum

The assembly 1 protein sequences deduced from the Fusarium graminearumpartial genome sequence (Broad Institute, MIT) were downloaded into aMicrosoft Word document. A search was done for sequences containing theGESAG motif, which is the conserved sequence around the active siteserine residue in Geotrichum candidum lipases (EMBL Accession Nos.U02622 and U02541) and homologs thereof. Several sequences were found tocontain the GESAG motif and those with a suitable size and additionalconserved residues close to the GESAG motif were aligned in full lengthwith the Geotrichum candidum lipases and homologs. The hypotheticalprotein FG01603.01 with a length of 591 amino acids was found to beclearly the best match. However, when the corresponding DNA wastranslated, it was found that the C-terminal sequence was more likely tobe GALVV rather than GALVDTNG as in FG01603.01.

Example 13 Fusarium graminearum Genomic DNA Extraction

Fusarium graminearum was grown in 100 ml of M400 medium in a baffledshake flask at 24° C. and 200 rpm. Mycelia were harvested by filtration,washed twice in TE, and frozen under liquid nitrogen. Frozen myceliawere ground, by mortar and pestle, to a fine powder, which wasresuspended in pH 8.0 buffer containing 10 mM Tris, 100 mM EDTA, 1%Triton X-100, 0.5 M guanidine-HCl, and 200 mM NaCl. DNase free RNase Awas added at a concentration of 20 mg/liter and the lysate was incubatedat 37° C. for 30 minutes. Cellular debris was removed by centrifugation,and DNA was isolated using a QIAGEN Maxi 500 column. The columns wereequilibrated in 10 ml of QBT, washed with 30 ml of QC, and eluted with15 ml of QF. DNA was precipitated in isopropanol, washed in 70% ethanol,and recovered by centrifugation. The DNA was resuspended in TE buffer.

Example 14 Cloning of a Fusarium graminearum Lipase Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify a Fusarium graminearum lipase gene from the genomic DNA preparedin Example 13.

Forward primer: (SEQ ID NO: 22) 5′-CACTGCTATCACCAACATGAGATTCTCTGG-3′Reverse primer: (SEQ ID NO: 23)5′-CCAACAAGGTATTTAATTAATCATACCACCAAAGC-3′Bold letters represent coding sequence.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. One μM of each of the primers above were used in aPCR reaction containing 200 ng of pSMO232 (Example 6), 1×PCR buffer with1.5 mM MgCl₂, 1 μl of dNTP mix (10 mM each), and 0.75 μl of DNApolymerase mix (3.5 U/μl) in a final volume of 50 μl. To amplify thefragment, an Eppendorf Mastercycler Thermocycler was programmed for 1cycle at 94° C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds,60° C. for 30 seconds, and 72° C. for 1.25 minutes; 15 cycles each at94° C. for 15 seconds, 60° C. for 30 seconds, and 72° C. for 1.25minutes plus a 5 second elongation at each successive cycle; 1 cycle at72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 0.8% agarose gel using TBEbuffer and a 1.8 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions.

The PCR fragment and pCR2.1-TOPO vector were ligated using conditionsspecified by the manufacturer to produce pSMO230 (FIG. 7). Two μl of thereaction was used to transform E. coli TOP10 One Shot competent cellsaccording to the manufacturer's instructions. A 2 μl volume of theligation mixture was added to the E. coli cells and incubated on ice for20 minutes. Subsequently, the cells were heat shocked for 30 seconds at42° C., and then placed on ice for 2 minutes. A 250 μl volume of SOCmedium was added to the cells and the mixture was incubated for 1 hourat 37° C. and 250 rpm. After the incubation the colonies were spread on2×YT plates supplemented with 100 μg of ampicillin per ml and incubatedat 37° C. overnight for selection of the plasmid. Sixteen colonies thatgrew on the plates were picked with a sterile toothpick and grownovernight at 37° C., 250 rpm in a 15 ml Falcon tube containing 3 ml ofLB medium supplemented with 100 μg of ampicillin per ml. An E. colitransformant containing a plasmid designated pSMO230 was detected byrestriction digestion and plasmid DNA was prepared using a BioRobot9600.

E. coli TOP10 One Shot cells containing pSMO23O were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30803, with a deposit date of Dec. 17, 2004.

Example 15 Characterization of the Fusarium graminearum Genomic SequenceEncoding Lipase

DNA sequencing of the Fusarium graminearum lipase gene from pSMO230 wasperformed with an Applied Biosystems Model 377 XL DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand all sequences were compared to each other with assistance ofPHRED/PHRAP software (University of Washington, Seattle, Wash.).

Gene models for the sequence were constructed based on the tfasty outputand alignment with a homologous lipase gene from Geotrichum candidum(SWALL P17577). A comparative alignment of amino acid sequences was madeusing the MAFFT method with iterative refinement and default parameters(Katoh et al., 2002, supra). The nucleotide sequence (SEQ ID NO: 3) anddeduced amino acid sequence (SEQ ID NO: 4) are shown in FIGS. 8A and 8B.The genomic fragment encodes a polypeptide of 588 amino acids. The % G+Ccontent of the gene is 52.5% and the mature protein coding region(nucleotides 73 to 1764) is 52.4%. Using the SignalP software program(Nielsen et al., 1997, supra), a signal peptide of 24 residues waspredicted. The predicted mature protein contains 564 amino acids with amolecular mass of 63 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Fusarium graminearum lipase geneshared 23% identity to the deduced amino acid sequence of a Geotrichumcandidum lipase gene (SWALL P17577).

Example 16 Construction of pEJG61

The Fusarium venenatum expression vector pEJG61 was generated bymodification of pSheB1 (U.S. Pat. No. 6,090,604). The modificationsincluded (a) changing the single Bsp LU11I site in pSheB1 bysite-directed mutagenesis (b) replacement of 930 bp of the Fusariumoxysporum trypsin promoter with 2.1 kilobases of the Fusarium venenatumglucoamylase promoter, and (c) introduction of a Bsp LU11I site afterthe Fusarium venenatum glucoamylase promoter.

Removal of the Bsp LU11I site within the pSheB1 sequence wasaccomplished using the QuikChange™ Site-Directed Mutagenesis Kit(Stratagene Cloning Systems, La Jolla, Calif.) according to themanufacturer's instruction with the following pairs of mutagenesisprimers:

(SEQ ID NO: 24) 5′-GCAGGAAAGAACAAGTGAGCAAAAGGC-3′ (SEQ ID NO: 25)5′-GCCTTTTGCTCACTTGTTCTTTCCTGC-3′This created pSheB1 intermediate 1.

Removal of 930 bp of the Fusarium oxysporum trypsin promoter wasaccomplished by digesting pSheB1 intermediate 1 (6,971 bp) with Stu Iand Pac I, subjecting the digest to electrophoresis on a 1% agarose gel,at 100 volts for one hour using TBE buffer, excising the 6,040 bp vectorfragment, and purifying the excised fragment with a Qiaquick GelPurification Kit (QIAGEN Inc., Valencia, Calif.). To introduce a new BspLU11I site, a linker was created using the following primers:

(SEQ ID NO: 26) 5′-dCCTACATGTTTAAT-3′ Bsp Lu11I (SEQ ID NO: 27)5′-dTAAACATGTAGG-3′Each primer (2 μg each) was heated to 70° C. for 10 minutes and thencooled to room temperature over an hour. This linker was ligated intothe Stu I-Pac I digested pSheB1 intermediate 1 vector fragment, creatingpSheBI intermediate 2.

A 2.1 kilobase fragment of Fusarium venenatum genomic DNA 5 prime of theglucoamylase coding region (glucoamylase promotor) was isolated by PCRof plasmid pFAMG (WO 00/56900) containing Fusarium venenatum genomic DNAencoding the entire coding region for Fusarium venenatum glucoamylaseand 3,950 bp of upstream sequence. The primers used for PCR follow:

(SEQ ID NO: 28) 5′-AGGCCTCACCCATCTCAACAC-3′ (SEQ ID NO: 29)5′-ACATGTTGGTGATAGCAGTGA-3′

The PCR reaction (50 μl) was composed of 200 ng of pFAMG, 200 μM dNTPs,1 μM of the above primers, 1× reaction buffer, and 2.6 units of ExpandHigh Fidelity enzyme mix. The reactions were incubated using a MJResearch Thermocycler (MJ Research, Inc., Boston, Mass.) programmed for1 cycle 1 at 94° C. for 2 minutes; 10 cycles each at 94° C. for 15seconds, 60° C. for 30 sec, and 72° C. for 2 minutes and 15 seconds; 15cycles each at 94° C. for 15 seconds, 60° C. for 30 seconds, and 72° C.for 2 minutes and 15 seconds with 5 seconds cycle elongation for eachsuccessive cycle; and 1 cycle at 72° C. for 7 minutes.

The PCR product was subjected to electrophoresis on a 1% agarose gel at100 volts for one hour using TBE buffer generating an expected band of2,108 bp. The 2,108 bp PCR product was excised from the agarose gel andpurified with a Qiaquick Gel Purification Kit. This fragment wasdigested with Stu I and Bsp LU11I, purified with a Qiaquick PurificationKit (QIAGEN Inc., Valencia, Calif.), and ligated into Stu I-Bsp LU11Idigested pSheBI intermediate 2 creating pEJG61 (FIG. 9).

Example 17 Construction of a Fusarium venenatum Expression VectorExpressing Fusarium graminearum Lipase Gene

The two synthetic oligonucleotide primers described in Example 14 weredesigned to PCR amplify the Fusarium graminearum lipase gene from thegenomic DNA prepared in Example 13.

The fragment of interest was amplified by PCR using the Herculase™Hotstart PCR System (Stratagene, La Jolla, Calif.). One μM of each ofthe primers above were used in a PCR reaction containing 20 ng ofFusarium graminerum genomic DNA, 1×PCR buffer (Stratagene, La Jolla,Calif.), 1 μl of dNTP mix (10 mM each), and 1.0 μl of DNA polymerase mix(5 U/μl; Stratagene, La Jolla, Calif.) in a final volume of 50 μl. Toamplify the fragment, an Eppendorf Mastercycler thermocycler wasprogrammed for 1 cycle at 94° C. for 2 minutes; 10 cycles each at 94° C.for 15 seconds, 60° C. for 30 seconds, and 72° C. for 1.25 minutes; 15cycles each at 94° C. for 15 seconds, 60° C. for 30 seconds, and 72° C.for 1.25 minutes plus a 5 second elongation at each successive cycle; 1cycle at 72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 0.8% agarose gel using TBEbuffer and the 1.8 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions.

The 1.8 kb PCR fragment containing the Fusarium graminearum lipase genewas cloned into pEJG61 using an InFusion Cloning Kit where the vectorwas digested with Bsp LU11I and Pac I. The digested vector was purifiedby gel electrophoresis using a 0.7% agarose gel with TBE buffer, and thePCR fragment was extracted using a QIAquick Gel Extraction Kit andpurified using a QIAquick PCR Purification Kit. The gene fragment andthe digested vector were ligated together in a reaction resulting in theexpression plasmid pSMO232 (FIG. 10). The ligation reaction (50 μl) wascomposed of 1× InFusion Buffer, 1×BSA, 1 μl of Infusion enzyme (diluted1:10), 100 ng of pEJG61 digested with Bsp LU11I and Pac I, and 50 ng ofthe Fusarium graminearum lipase gene purified PCR product. The reactionwas incubated at room temperature for 30 minutes. Two μl of the reactionwas used to transform E. coli SoloPack Gold supercompetent cellsaccording to the manufacturer's instructions. One μl ofβ-mercaptoethanol was added to the competent cells, and incubated on icefor 10 minutes. A 2 μl volume of the ligation mixture was then added tothe E. coli cells and incubated on ice for 30 minutes. Subsequently, thecells were heat shocked for 60 seconds at 54° C., and then placed on icefor 2 minutes. A 150 μl volume of NZY⁺ medium at 42° C. was added to thecells and the mixture was incubated for 1 hour at 37° C. and 250 rpm.After the incubation the colonies were spread on 2×YT platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight for selection of the plasmid. Nine colonies that grew on theplates were picked with a sterile toothpick and grown overnight at 37°C., 250 rpm in a 15 ml Falcon tube containing 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. An E. coli transformantcontaining pSMO232 was detected by restriction digestion and plasmid DNAwas prepared using a QIAGEN BioRobot 9600.

Example 18 Expression of the Fusarium graminearum Lipase Gene inFusarium venenatum WTY842-1-11

Spores of Fusarium venenatum strain WTY842-1-11 (Δtrichodiene synthasegene, amdS⁺) a mutant of Fusarium strain A3/5 (NRRL 30747 or ATCC 20334)(Wiebe et al., 1992, Mycological Research 96: 555-562; Wiebe et al.,1991, Mycological Research 95: 1284-1288; Wiebe et al., 1991,Mycological Research 96: 555-562), were generated by inoculating a flaskcontaining 100 ml of RA sporulation medium with 8 plugs from an agarplate and incubated at 27° C., 150 rpm for 24 hours, then 24 hours, 150rpm at 22° C. Spores were harvested by filtering culture throughMIRACLOTH™ (Calbiochem, La Jolla, Calif.) onto a Nalgene 0.2 μm filter.Wash spores twice with sterile distilled water, resuspended in a smallvolume of water, and then counted using a hemocytometer.

Protoplasts were prepared by inoculating 100 ml of YPG medium with 2×10⁸spores of Fusarium venenatum WTY842-1-11 and incubating for 15 hours at18° C. and 150 rpm. The culture was filtered through MIRACLOTH™, washedonce with sterile distilled water and once with 1 M MgSO₄ andresuspended in 40 ml of 5 mg/ml of NOVOZYME 234™ (Novozymes A/S,Bagsværd, Denmark) in 1 M MgSO₄. Cultures were incubated at 29° C. and90 rpm until protoplasts formed. A volume of 30 ml of 1 M sorbitol wasadded to the protoplast digest and the mixture was centrifuged at 1500rpm for 10 minutes. The pellet was resuspended, washed twice with 1 Msorbitol, and centrifuged at 1500 rpm for 10 minutes to pellet theprotoplasts. Protoplasts were counted with a hemocytometer andresuspended in an 8:2:0.1 solution of STC:SPTC:DMSO to a finalconcentration of 5×10⁷ protoplasts/ml. The protoplasts were stored at−80° C., after controlled-rate freezing in a Nalgene Cryo 1° C. FreezingContainer.

Two ml of protoplast suspension were added to 20 μg of pSMO232 and 250μg of heparin in a 50 ml Falcon tube, mixed and incubated on ice for 30minutes. Two hundred μl of SPTC was mixed gently into the protoplastsuspension and incubation was continued at room temperature for 10minutes. Twenty ml of SPTC was mixed gently into the protoplastsuspension and incubation was continued at room temperature for 10minutes. A 350 ml volume of molten Vogel's NO₃ regeneration low-meltmedium (cooled to 50° C.) was mixed with the protoplasts and then 35 mlwere plated onto 100 mm Petri plate containing 35 ml of the identicalmedium plus 12 mg of BASTA™ per ml. Incubation was continued at roomtemperature for 6 days. After 6 days, 29 transformants were apparent. Amycelial fragment from the edge of each transformant was transferred toplates containing Vogel's NO₃ regeneration low-melt medium plus BASTA™(6 mg/ml) medium. The plate was sealed in a plastic bag to maintainmoisture and incubated approximately one week at room temperature.

One cm² of spore growth was inoculated separately into 25 ml of MY400medium in 125 ml plastic shake flasks and incubated at 28° C., 250 rpm.Three and five days after incubation, culture supernatants were removedfor lipase assay and SDS-PAGE analysis.

Lipase activity was determined as described in Example 12. The lipaseassay results indicated that at 3 days several of the transformantsproduced lipase activity above that of the untransformed control.SDS-PAGE analysis (BioRad Criterion 10-20% SDS-PAGE) of 10 μl of thesupernatants showed a band at approximately 61 kDa.

Example 19 Determination of Substrate Specificity of RecombinantFusarium graminearum Lipase

The substrate specificity of the Fusarium graminearum lipase wasdetermined using a panel screen composed of 4-nitrophenol (PNP) lipasesubstrates as described in Example 9.

The results for the panel screen are shown in Table 4.

TABLE 4 Ratio of PNP-substrates according to Table 1, Example 9, where:P = Palmitate, D = Decanoate, V = Valerate and B = Butyrate Longchain/Short chain at pH 7.5 P/V D/V P/B D/B F. graminearum 0.769 0.7720.398 0.399 lipase LIPEX ™ 0.997 1.944 2.122 4.080 pH Ratios; (−) = 0.2%Triton data 9.5P/7.0P 9.5D/7.0D 9.5P−/7.5P− F. graminearum 0.138 0.393Not Tested lipase LIPEX ™ 2.072 2.574 2.251 No or Low Triton (−)compared to maximum Triton (+) V7.5−/+ D9.5+/− P7.5+/− P9.5+/− F.graminearum Not Not Not Tested Not Tested lipase Tested Tested LIPEX ™7.108 5.110 9.331 8.166 Fusarium graminearum lipase (Fusarium venenatumWTY-842-1-11 expressing Fusarium graminearum lipase 1) test dataconsists of the average of 2 assay results; and LIPEX ™ data consists ofthe average a minimum of 80 assay results.

In comparing the panel screen results of LIPEX™ and the Fusariumgraminearum lipase the following observations were made:

1. The ratio of activities on PNP-palmitate at pH 9.5 versus pH 7 is16-fold lower for the Fusarium graminearum lipase versus LIPEX™suggesting that the Fusarium graminearum lipase has much lower activityat pH 9.5 versus LIPEX™ or it has a much higher activity at pH 7.0 thanLIPEX™ or a combination of these two.

2. The ratios of PN, DN, and D/B are also quite different for theFusarium graminearum lipase versus LIPEX™ suggesting there is some acylchange length specificity differences between the two lipases.

Example 20 Identification of Lipase Genes in the Partial GenomicSequence of Magnaporthe grisea

A tfasty search (Pearson, W. R., 1999, supra) of the Magnaporthe griseapartial genome sequence (The Institute for Genomic Research, Rockville,Md.) was carried out using as query a lipase sequence from Geotrichumcandidum (SWALL P17573). Several genes were identified as putativelipases based upon a high degree of similarity to the query sequence atthe amino acid level. Two genomic regions of approximately 1400 bp and110 bp with greater than 33% identity to the query sequence at the aminoacid level were chosen for further study. Gene models for the putativelipase genes were predicted based on homology to the Geotrichum candidumlipase as well as conserved sequences present at the 5′ and 3′ ends offungal introns.

Example 21 Magnaporthe grisea Genomic DNA Extraction

Four hundred μl of Magnaporthe grisea (FGSC 8958, Fungal Genetics StockCenter) spores were grown in 50 ml of CM medium in a baffled shake flaskat 25° C. and 250 rpm for 4 days. Genomic DNA was then extracted fromthe mycelia using a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia,Calif.) according to manufacturer's instructions.

Example 22 Cloning of the Magnaporthe grisea Lipase 1 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify a Magnaporthe grisea gene encoding a lipase 1 from the genomicDNA prepared in Example 21.

Forward primer: (SEQ ID NO: 30)5′-ACACAACTGGCCATGAAAGCCTCCATTCTTTCGGCT-3′ Reverse primer:(SEQ ID NO: 31) 5′-AGTCACCTCTAGTTAATTAATTAAATGTAAAAGCTGAGGA-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a (see Example 6).

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. One μM of each of the primers above were used in aPCR reaction containing 300 ng of Magnaporthe grisea genomic DNA, 1×PCRbuffer with 1.5 mM MgCl₂, 1 μl of dNTP mix (10 mM each), and 0.75 μl ofDNA polymerase mix (3.5 U/μl) in a final volume of 50 μl. To amplify thefragment, an Eppendorf Mastercycler thermocycler was programmed for 1cycle at 94° C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds,59.2° C. for 30 seconds, and 72° C. for 1.25 minutes; 15 cycles each at94° C. for 15 seconds, 59.2° C. for 30 seconds, and 72° C. for 1.25minutes plus a 5 second elongation at each successive cycle; 1 cycle at72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 0.7% agarose gel using TBEbuffer and the 2 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions. The PCRfragment and pCR2.1-TOPO were ligated using conditions specified by themanufacturer resulting in plasmid pJLin175 (FIG. 11).

Two μl of the reaction was used to transform E. coli TOP10 One Shotcompetent cells according to the manufacturer's instructions. A 2 μlvolume of the ligation mixture was added to the E. coli cells andincubated on ice for 5 minutes. Subsequently, the cells were heatshocked for 30 seconds at 42° C., and then placed on ice for 2 minutes.A 250 μl volume of SOC medium was added to the cells and the mixture wasincubated for 1 hour at 37° C. and 250 rpm. After the incubation thecolonies were spread on 2×YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Twelve colonies that grew on the plates were picked with asterile toothpick and grown overnight at 37° C., 250 rpm in a 15 mlFalcon tube containing 3 ml of LB medium supplemented with 100 μg ofampicillin per ml. An E. coli transformant containing the pJLin175 wasdetected by restriction digestion and plasmid DNA was prepared using aBioRobot 9600.

E. coli TOP 10 One Shot cells containing pJLin175 were deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30783, with a deposit date of Oct. 12, 2004.

Example 23 Characterization of the Magnaporthe grisea Genomic SequenceEncoding Lipase 1

DNA sequencing of the Magnaporthe grisea lipase 1 gene from pJLin175 wasperformed with an Applied Biosystems Model 377 XL DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand all sequences were compared to each other with assistance ofPHRED/PHRAP software (University of Washington, Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to theGeotrichum candidum lipase as well as conserved sequences present at the5′ and 3′ ends of fungal introns. A comparative alignment of amino acidsequences was made using the MAFFT method with iterative refinement anddefault parameters (Katoh et al., 2002, supra). The nucleotide sequence(SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO: 6) are shownin FIGS. 12A and 12B. The genomic fragment encodes a polypeptide of 598amino acids. The % G+C content of the gene is 55.5% and of the matureprotein coding region (nucleotides 61 to 1922 of SEQ ID NO: 5) is 55.5%.Using the SignalP software program (Nielsen et al., 1997, supra), asignal peptide of 20 residues was predicted. The predicted matureprotein contains 578 amino acids with a molecular mass of 64.3 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Magnaporthe grisea lipase 1 geneshared 33% identity to the deduced amino acid sequence of a Geotrichumcandidum lipase gene (SWALL P17573).

Example 24 Cloning of a Magnaporthe grisea Lipase 2 Gene

Several synthetic oligonucleotide primers shown below were designedbased on the predicted sequence to PCR amplify another Magnaporthegrisea gene encoding a lipase 2 from the genomic DNA.

Forward primers: (SEQ ID NO: 32) 5′-ATTTTCCCGGCTCGACGCTTCTGT-3′(SEQ ID NO: 33) 5′-TCATGATGCGTCAATCCATCTTCCAGT′-3 (SEQ ID NO: 34)5′-GGACGAGTTCGTCGCCATGGTGCAGC′-3 Reverse primers: (SEQ ID NO: 35)5′-GCTGCACCATGGCGACGAACTCGTCC′-3 (SEQ ID NO: 36)5′-TTTAATTAATTAGTACCCGCAAATCCATAACAACAAC-3′Bold letters represent coding sequence. The remaining sequence washomologous to the enzyme restriction insertion sites of pBM120a.

The fragments of interest were amplified by PCR using the Herculase™Hotstart DNA polymerase system. The First Fragment (Fragment 1) wasgenerated using 50 ng of primer SEQ ID NO: 32 and 50 ng of primer SEQ IDNO: 36 in a PCR reaction (50 μl) containing 300 ng of Magnaporthe griseagenomic DNA, 1× Herculase PCR buffer (Stratagene, La Jolla, Calif.)with, 1 μl of dNTP mix (10 mM each), 2 μl DMSO, and 2.5 units ofHerculase™ Hotstart DNA polymerase (Stratagene, La Jolla, Calif.).Fragment 2 was amplified using 5 μl of Fragment 1 (PCR reaction) astemplate with 50 ng of primer SEQ ID NO: 32 and 50 ng of primer SEQ IDNO: 35. Fragment 3 was amplified using 5 μl of Fragment 1 (PCR reaction)as template with 50 ng each primers SEQ ID NO: 34 and SEQ ID NO: 36. ThePeltien thermal cycler (MJ Research Inc., Watertown, Mass.) wasprogrammed for 1 cycle at 92° C. for 2 minutes; 30 cycles each at 92° C.for 1 minute, 51° C. for 1 minute, 72° C. for 1 minute; 1 cycle at 72°C. for 10 minutes; and a 4° C. hold.

The reaction products were visualized on a 1% agarose gel using TAEbuffer and the product bands were purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions. The PCRFragment 1 and pCR2.1-TOPO were ligated using conditions specified bythe manufacturer resulting in plasmid pCrAm140 (FIG. 13)

Two μl of the reaction was used to transform E. coli TOP10 One Shotcompetent cells according to the manufacturer's instructions. A 2 μlvolume of the ligation mixture was added to the E. coli cells andincubated on ice for 5 minutes. Subsequently, the cells were heatshocked for 30 seconds at 42° C., and then placed on ice for 2 minutes.A 250 μl volume of SOC medium was added to the cells and the mixture wasincubated for 1 hour at 37° C. and 250 rpm. After the incubation thecolonies were spread on 2×YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Twenty colonies that grew on the plates were picked with asterile toothpick and grown overnight at 37° C., 250 rpm in a 15 mlFalcon tube containing 3 ml of LB medium supplemented with 100 μg ofampicillin per ml. An E. coli transformant containing the pCrAm140 wasdetected by restriction digestion and plasmid DNA was prepared using aBioRobot 9600.

E. coli TOP 10 One Shot cells containing pCrAm140 were deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30788 on Dec. 1, 2004.

Example 25 Characterization of the Margnaporthe grisea Genomic SequenceEncoding Lipase 2

DNA sequencing of the Magnaporthe grisea lipase 2 gene from pCrAm140 wasperformed with an Applied Biosystems Model 377 XL DNA Sequencer usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand all sequences were compared to each other with assistance ofPHRED/PHRAP software (University of Washington, Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to theGeotrichum candidum lipase as well as conserved sequences present at the5′ and 3′ ends of fungal introns. A comparative alignment of amino acidsequences was made using the MAFFT method with iterative refinement anddefault parameters (Katoh et al., 2002, supra). The nucleotide sequence(SEQ ID NO: 7) and deduced amino acid sequence (SEQ ID NO: 8) are shownin FIGS. 14A and 14B. The genomic fragment encodes a polypeptide of 534amino acids. The % G+C content of the gene is 66.1% and of the matureprotein coding region (nucleotides 55 to 1602 of SEQ ID NO: 7) is 66.1%.Using the SignalP software program (Nielsen et al., 1997, supra, asignal peptide of 18 residues was predicted. The predicted matureprotein contains 516 amino acids with a molecular mass of 55.5 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Magnaporthe grisea lipase 2 geneshared 33% identity to the deduced amino acid sequence of a Geotrichumcandidum lipase gene (SWALL P17573).

Example 26 Identification of Lipase Genes in the Partial GenomicSequence of Neurospora crassa

A tfasty search (Pearson, W. R., 1999, supra) of the Neurospora crassapartial genome sequence (Broad Institute, MIT) was carried out using asquery a lipase sequence from Geotrichum candidum (SWALL P17577). A genewas identified as putative lipases based upon a high degree ofsimilarity to the query sequence at the amino acid level. One genomicregion of approximately 1200 bp with greater than 38% identity to thequery sequence at the amino acid level was chosen for further study.Gene models for the putative lipase genes were predicted based onhomology to the Geotrichim candidum lipase 1 gene as well as conservedsequences present at the 5′ and 3′ ends of fungal introns.

Example 27 Neurospora crassa Genomic DNA Extraction

Four hundred μl of Neurospora crassa (FGSC 2489, Fungal Genetics StockCenter) spores were grown in 50 ml of CM medium in a baffled shake flaskat 25° C. and 250 rpm for 4 days. Genomic DNA was then extracted fromthe mycelia using the following method. YEG medium (100 ml) supplementedwith 1% additional glucose was inoculated from a plate of PDA plate andincubated for 2 days at 34° C. Mycelia were collected by filtration on aWhatmann #1 filter, frozen in liquid nitrogen, and ground to a powder ina mortar and pestle on dry ice. One-fourth of this material wasincubated for 60 minutes at 60° C. with 20 ml of TE containing 20 mMCAPS-NaOH pH 11.0 buffer and 1% lithium dodecyl sulfate. This wasextracted with an equal volume of phenol:chloroform:isoamyl alcohol(25:24:1) on a rotating wheel for 60 minutes at 37° C., centrifuged at2500 rpm for 5 minutes, and the aqueous phase re-extracted in the samefashion. The aqueous phase was brought to 2.5 M ammonium acetate andfrozen. It was thawed, the nucleic acids precipitated with 0.7 volisopropanol, and the precipitate collected by centrifugation at 15,000×gfor 20 minutes. The pellet was rinsed twice with 70% ethanol, air dried,and dissolved in 1.0 ml of 0.1×TE. RNase was added to 100 μg per ml andthe tube incubated for 30 minutes at room temperature. The solution wasbrought to 2 M ammonium acetate and DNA precipitated by addition of 2volumes of ethanol and collected by centrifugation at 13,000×g for 20minutes at room temperature. The pellet was rinsed twice with 70%ethanol, air-dried, and dissolved in 0.75 ml of 0.1×TE.

Example 28 Cloning of a Neurospora crassa Lipase Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify a Neurospora crassa lipase gene from the genomic DNA prepared inExample 27.

Forward primer: (SEQ ID NO: 37)5′-ACACAACTGGCCATGAAGGGCTTTTCCAACGCTCTCCTCG-3′ Reverse primer:(SEQ ID NO: 38) 5′-AGTCACCTCTAGTTAATTAATTAGATGTGAAGAGCATCAAGATTAG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System (Roche Diagnostics, Mannheim, Germany). Fiftypicomoles of each of the primers above were used in a PCR reactioncontaining 550 ng of Neurospora Crassa genomic DNA. The PCRamplification reaction mixture also contained 1×PCR buffer with 1.5 mMMgCl2, 1 μl of dNTP mix (10 mM each), and 0.75 μl DNA polymerase mix(3.5 U/μl) in a final volume of 50 μl. An Eppendorf Mastercyclerthermocycler was used to amplify the fragment programmed for 1 cycle at94° C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds, 62° C.for 30 seconds, and 72° C. for 2 minutes; 15 cycles each at 94° C. for15 seconds, 62° C. for 30 seconds, and 72° C. for 2 minutes plus a 5second elongation at each successive cycle; 1 cycle at 72° C. for 7minutes; and a 10° C. hold.

The reaction product was visualized on a 1.0% agarose gel using TBEbuffer and the 1.8 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions.

The 1.8 kb PCR product was then cloned into pCR2.1-TOPO (Invitrogen,Carlsbad, Calif.) according to manufacturer's instructions to producepBM134b (FIG. 15). Two μl of the reaction was used to transform E. coliTOP10 One Shot competent cells. An E. coli transformant containingpBM134b was detected by restriction digestion and plasmid DNA wasprepared using a QIAGEN BioRobot 9600.

E. coli TOP 10 One Shot cells containing pBM134b were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30786, with a deposit date of Nov. 17, 2004.

Example 29 Characterization of the Neurospora crassa Genomic SequenceEncoding Lipase

DNA sequencing of the Neurospora crassa lipase gene from pBM134b wasperformed with a Perkin-Elmer Applied Biosystems Model 377 XL AutomatedDNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,Calif.) using dye-terminator chemistry (Giesecke et al., 1992, Journalof Virology Methods 38: 47-60) and primer walking strategy. Nucleotidesequence data were scrutinized for quality and all sequences wereanalyzed with assistance of ContigExpress software (Informax, Inc.,Bethesda, Md.).

Gene models for the lipase gene were predicted based on homology to theGeotrichum candidum lipase 1 as well as conserved sequences present atthe 5′ and 3′ ends of fungal introns. A comparative alignment of aminoacid sequences was made using the MAFFT method with iterative refinementand default parameters (Katoh et al., 2002, Nucleic Acids Research 30:3059). The genomic coding sequence (SEQ ID NO: 9) and deduced amino acidsequence (SEQ ID NO: 10) are shown in FIGS. 16A and 16B. The genomicfragment encodes a polypeptide of 578 amino acids, interrupted by 1intron of 55 bp. The % G+C content of the gene is 56.19% and of themature protein coding region (nucleotides 64 to 1789 of SEQ ID NO: 9) is56.88%. Using the SignalP software program (Nielsen et al., 1997,Protein Engineering 10:1-6), a signal peptide of 21 residues waspredicted. The predicted mature protein contains 557 amino acids with amolecular mass of 59.97 kDa.

A comparative alignment of lipase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Neurospora crassa lipase gene shared31% identity to the deduced amino acid sequences of the Geotrichumcandidum lipase 1 gene (SWALL P17577).

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli pJLin173 NRRL B-30782Oct. 12, 2004 E. coli pSMO230 NRRL B-30803 Dec. 17, 2004 E. colipJLin175 NRRL B-30783 Sep. 13, 2004 E. coli pCrAm140 NRRL B-30788 Dec.1, 2004 E. coli pBM134b NRRL B-30786 Nov. 17, 2004

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. An isolated polynucleotide encoding a polypeptidehaving lipase activity, selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having lipase activity comprisingan amino acid sequence having at least 90% identity with the maturepolypeptide of SEQ ID NO: 6; (b) a polynucleotide encoding a polypeptidehaving lipase activity comprising a nucleotide sequence which hybridizesunder at least high stringency conditions with the mature polypeptidecoding sequence of SEQ ID NO: 5 or its full-length complementary strand,wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide and a wash three times,each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; and (c) apolynucleotide encoding a polypeptide having lipase activity comprisinga nucleotide sequence having at least 90% identity with the maturepolypeptide coding sequence of SEQ ID NO:
 5. 2. The isolatedpolynucleotide of claim 1, wherein the polypeptide comprises an aminoacid sequence having at least 90% identity with the mature polypeptideof SEQ ID NO:
 6. 3. The isolated polynucleotide of claim 2, wherein thepolypeptide comprises an amino acid sequence having at least 95%identity with the mature polypeptide of SEQ ID NO:
 6. 4. The isolatedpolynucleotide of claim 3, wherein the polypeptide comprises an aminoacid sequence having at least 97% identity with the mature polypeptideof SEQ ID NO:
 6. 5. The polynucleotide of claim 1, wherein thepolypeptide comprises or consists of the amino acid sequence of SEQ IDNO: 6 or the mature polypeptide thereof, or a fragment thereof havinglipase activity.
 6. The isolated polynucleotide of claim 1, whichcomprises a nucleotide sequence which hybridizes under at least highstringency conditions with the mature polypeptide coding sequence of SEQID NO: 5 or its full-length complementary strand, wherein highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide and a wash three times, each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.
 7. The isolated polynucleotide of claim6, which comprises a nucleotide sequence which hybridizes under at leastvery high stringency conditions with the mature polypeptide codingsequence of SEQ ID NO: 5 or its full-length complementary strand,wherein very high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide and a wash three times,each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.
 8. Thepolynucleotide of claim 1, which comprises a nucleotide sequence havingat least 90% identity with the mature polypeptide coding sequence of SEQID NO:
 5. 9. The polynucleotide of claim 8, which comprises a nucleotidesequence having at least 95% identity with the mature polypeptide codingsequence of SEQ ID NO:
 5. 10. The polynucleotide of claim 9, whichcomprises a nucleotide sequence having at least 97% identity with themature polypeptide coding sequence of SEQ ID NO:
 5. 11. Thepolynucleotide of claim 1, which comprises or consists of the nucleotidesequence of SEQ ID NO: 5 or the mature polypeptide coding sequencethereof, or a subsequence thereof that encodes a fragment having lipaseactivity.
 12. The polynucleotide of claim 1, which is encoded by thepolynucleotide contained in plasmid pJLin175 which is contained in E.coli NRRL B-30783.
 13. The polynucleotide of claim 1, wherein the maturepolypeptide is amino acids 21 to 598 of SEQ ID NO:
 6. 14. Thepolynucleotide of claim 1, wherein the mature polypeptide codingsequence is nucleotides 61 to 1922 of SEQ ID NO:
 5. 15. A nucleic acidconstruct or expression vector comprising the polynucleotide of claim 1operably linked to one or more control sequences that direct theproduction of the polypeptide in an expression host.
 16. A recombinanthost cell comprising the polynucleotide of claim 1 operably linked toone or more control sequences that direct the production of thepolypeptide.
 17. A method for producing a polypeptide having lipaseactivity, comprising: (a) cultivating the host cell of claim 16 underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 18. A method for producing a polypeptidehaving lipase activity, comprising: (a) cultivating a transgenic plantor a plant cell comprising the polynucleotide of claim 1 underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 19. A transgenic plant, plant part or plantcell, which has been transformed with the polynucleotide of claim 1.